Enzymes catalyze biochemical reactions and play a major role in performing and controlling most life processes. Therefore, a detailed understanding of biological systems requires an understanding of the action of the corresponding enzymes. The importance of such an understanding is highlighted by the fact that many diseases can be controlled by developing drugs that block the action of enzymes in the crucial biological pathways of the pathogens that cause these diseases. It is also possible, at least in principle, to develop drugs that restore the activity of defective enzymes that are involved in devastating diseases. Another important development has been the emergence of the field of enzyme design, with promising advances in directed evolution and in computer aided design. However, this progress has not yet led to designer enzymes that can rival native enzymes. Thus, the potential of this important field can be enhanced in a major way by computational approaches that actually determine the activation barriers of the reactions that are being catalyzed. During previous grant periods, we developed refined and applied powerful methods for simulating reactions in enzymes and examined their performance. Using these methods helped us to quantify key catalytic factors and brought us to a stage where we can make significant contributions to the new frontiers of enzyme design and the exploration of catalytic landscapes. Here, we propose the following projects: (i) We will invest major effort into computer-aided enzyme design by: (a) advancing the use of the EVB as a quantitative tool in the final stage of enzyme design;(b) developing coarse grained approaches for the different screening stages, and (c) using our approaches in actual enzyme design projects, including changing the action of promiscuous enzymes, improving available designer enzymes and helping in the design of new enzymes. (ii) We will continue to develop ab initio-free energy perturbation approaches to a level where they can be used effectively in studies of enzymatic reactions. This will include: (a) improving the use of EVB reference potentials for QM(ai)/MM free energy simulations;(b) developing and refining our accelerated QM/MM approach with average potentials and a Langevin dynamics based potential of mean force, and (c) refining the use of the CDFT method in studies of metalloenzymes and in free energy mapping. (iii) We will quantify the relationship between folding and stability by advancing the following projects: (a) exploring the relationship between the pre-organization of the active sites and the local stability of the protein;(b) exploring the relationship between thermostability and catalysis, and (c) using a simplified model to evaluate the total stability and the corresponding chemical activation free energy. (iv) We will conduct studies of several important classes of enzymatic reactions. (v) We will continue with the systematic examination of different non-electrostatic catalytic proposals.